Method for mass production of plant-derived extracellular vesicles, and cosmetic composition thereof

Abstract

The present invention relates to a method for mass production of plant-derived extracellular vesicles, and a cosmetic composition capable of providing beneficial effects to the skin, comprising plant-derived (particularly, red cabbage-derived) extracellular vesicles as an active ingredient. More specifically, the present invention relates to: a method for isolating plant-derived extracellular vesicles; and a cosmetic composition comprising plant-derived extracellular vesicles obtained by the isolation method, the method sequentially comprising the steps of: preparing plant juice; centrifuging the plant juice and then collecting the supernatant; performing ultrafiltration of the supernatant to obtain a filtrate; and purifying the filtrate. The present invention enables the efficient isolation and purification of plant-derived extracellular vesicles with high purity and high quality, and plant-derived extracellular vesicles prepared by the method of the present invention are highly cell-permeable, and, when delivered, exhibit beneficial effects, such as antioxidant, anti-inflammatory, and skin-soothing effects, on the skin, and thus can be effectively used in a cosmetic composition.

Description

Method for mass production of plant-derived extracellular vesicles, and cosmetic composition related thereto

[0001] The present invention relates to a method for mass-producing plant-derived extracellular vesicles and a cosmetic composition capable of providing beneficial effects to the skin using plant-derived (particularly red cabbage-derived) extracellular vesicles as an active ingredient.

[0002] Extracellular Vesicles (EVs) are a general term for vesicles with a double phospholipid membrane structure that are secreted from the cells of an organism. The size of extracellular vesicles ranges from 20–30 nm to less than 1 µm in terms of particle size, and exosomes, which are representative extracellular vesicles, are known to have a particle size of 50–300 nm. Extracellular vesicles are also classified into types such as exosomes, ectosomes, and microvesicles depending on their size and the method of their formation.

[0003] Recently, it has been revealed that extracellular vesicles contain various types of physiologically active substances, such as proteins, lipids, and nucleic acids, and that they play roles as intercellular signal messengers, reaction mediators, and immunoreactants.

[0004] In other words, the extracellular vesicle is a transmembrane vesicle characterized by containing specific molecules such as proteins, lipids, metabolites, and nucleic acids derived from the parent cell, and by surrounding and protecting them with a lipid bilayer, it performs the role of delivering substances to other cells after secretion.

[0005] Recently, drug and vaccine delivery technologies utilizing these extracellular vesicles are being developed, based on their role as intercellular signaling molecules through mass transfer functions.

[0006] In particular, extracellular vesicles derived from plant cells have been reported to have lower cytotoxicity than other extracellular vesicles derived from animal or microbial cells, and they are also price-competitive in terms of supplying raw materials for the production and manufacture of extracellular vesicles.

[0007] However, unlike animal cells, plant cells possess cell walls, which necessitates somewhat complex processes such as cell wall crushing or fragmentation to obtain extracellular vesicles. Furthermore, since the yield of extracellular vesicles is relatively low, there is a need for new methods to improve it. Additionally, the process of isolating extracellular vesicles from tissues is limited in quantity and complex, posing a problem where high-purity purification is difficult using existing methods.

[0008] Looking at the prior art regarding this, the generally used ultra-high-speed centrifugation method has problems such as relatively high impurity concentrations and potential physical damage to extracellular vesicles due to strong centrifugal force, and the polymer precipitation method also has very high impurity concentrations and potential contamination problems due to the use of PEG (polyethylene glycol), and the separation method using size exclusion chromatography (SEC) takes excessive time, which limits its commercial application, especially mass production.

[0009] Furthermore, despite the advantage of plant-derived extracellular vesicles having very few side effect issues, research on the biological functions and applications of plant-derived extracellular vesicles used as food has not yet been systematically conducted, and there are still many unknown areas regarding the various efficacy of plant-derived extracellular vesicles as topical cosmetic agents.

[0010] For example, prior art regarding plant-derived extracellular vesicles can be understood by exemplarily referring to KR 10-2527607 B1 (Patent Document 1), which discloses a method for producing stable extracellular vesicles from plants in high yield and a composition containing said extracellular vesicles as an active ingredient (thereby, the entire contents of Patent Document 1 are incorporated and cited as prior art in the contents of this specification), but this document only reveals the degree of physiological effect of reducing NO production of plant-derived extracellular vesicles.

[0011] Therefore, further research is needed on manufacturing methods suitable for the mass production of plant-derived extracellular vesicles and on their physiological efficacy.

[0012] [Prior Art Literature]

[0013] [Patent Literature]

[0014] (Patent Document 1) KR 10-2527607 B1 (2023.04.26.)

[0015] The inventors have completed the present invention by conducting continuous research to develop an efficient method for obtaining plant-derived extracellular vesicles, specifically plant-derived extracellular vesicles derived from red cabbage. As a result, they discovered that by applying a process differentiated from conventional purification methods, it is possible to commercially obtain high-purity, high-quality red cabbage-derived extracellular vesicles in large quantities while preserving their original pharmacological activity and drug delivery capabilities, thereby shortening processing time and preventing physical damage to the extracellular vesicles. Accordingly, the objective of the present invention is to provide a method for efficiently isolating plant-derived extracellular vesicles.

[0016] In addition, the inventors have confirmed that these red cabbage-derived extracellular vesicles have various beneficial effects on the skin, such as skin soothing efficacy, which inhibits NO, IL-6, and IL-1β produced in excess by LPS containing these red cabbage-derived extracellular vesicles as active ingredients. Therefore, another objective of the present invention is for a cosmetic composition containing red cabbage-derived extracellular vesicles to provide skin soothing efficacy.

[0017] The present invention has been devised to solve the problems of the aforementioned prior art, and

[0018] Step of preparing plant juice;

[0019] A step of collecting the supernatant after centrifuging the above extract;

[0020] A step of obtaining a filtrate by performing ultrafiltration on the above supernatant; and

[0021] A method for isolating plant-derived extracellular vesicles is provided, which sequentially includes the step of purifying the above-mentioned filtrate.

[0022] In addition, the present invention provides a method for isolating plant-derived extracellular vesicles, characterized in that the centrifugation is performed at 4,000 to 30,000 g.

[0023] In addition, the present invention provides a method for isolating plant-derived extracellular vesicles, characterized in that the step of collecting the supernatant after centrifuging the extract is repeated two or more times, and the gravitational acceleration during centrifugation is gradually increased.

[0024] In addition, the present invention provides a method for isolating plant-derived extracellular vesicles, characterized in that the first centrifugation is performed at 4,000 to 10,000 g and the second centrifugation is performed at 15,000 to 30,000 g.

[0025] In addition, the present invention provides a method for isolating plant-derived extracellular vesicles, characterized in that the ultrafiltration has a cut-off value of 50 to 150 kDa.

[0026] In addition, the present invention provides a method for isolating plant-derived extracellular vesicles, characterized in that the purification step uses activated carbon.

[0027] In addition, the present invention provides a method for isolating plant-derived extracellular vesicles, characterized in that the plant is red cabbage.

[0028] In addition, a cosmetic composition comprising a plant-derived extracellular vesicle obtained by the separation method of the present invention is provided.

[0029] In addition, in the composition of the present invention, the extracellular vesicle is 1 x 10 per unit volume of 1 mL of the composition, based on the volume of the composition. 8 Pieces ~ 1 x 10 13 A cosmetic composition is provided that is characterized by containing particles.

[0030] In addition, the present invention provides a cosmetic composition characterized by containing extracellular vesicles derived from red cabbage as an active ingredient and having antioxidant efficacy.

[0031] In addition, the present invention provides a cosmetic composition characterized by containing extracellular vesicles derived from red cabbage as an active ingredient and having anti-inflammatory efficacy.

[0032] In addition, the present invention provides a cosmetic composition characterized by containing extracellular vesicles derived from red cabbage as an active ingredient and having a skin soothing effect.

[0033] The present invention enables the efficient separation and purification of plant-derived extracellular vesicles with high purity and quality. The plant-derived extracellular vesicles produced by the method of the present invention allow for good intracellular penetration, and since the delivered extracellular vesicles have beneficial effects on the skin, such as antioxidant, anti-inflammatory, and skin soothing effects, they can be usefully utilized in cosmetic compositions.

[0034] Figures 1 and 2 are the results of a nanoparticle tracking analyzer (NTA) measurement of extracellular vesicles derived from red cabbage according to an embodiment of the present invention.

[0035] Figure 3 is the result of a test on the NO generation inhibition effect of the cosmetic composition of the embodiment of the present invention.

[0036] FIGS. 4 and FIGS. 5 are the test results of the inhibitory effect of the cosmetic composition of the embodiment of the present invention on the production of IL-6 and IL-1β.

[0037] Figure 6 is the result of a clinical test on the soothing effect of the cosmetic composition of the present invention on skin that has been physically stimulated.

[0038] The present invention will be described in detail below.

[0039]

[0040] To achieve the above objective, the present invention aims to provide an efficient method for obtaining extracellular vesicles derived from red cabbage, and also to provide a cosmetic composition having antioxidant, anti-inflammatory, or skin-soothing effects.

[0041] The above red cabbage-derived extracellular vesicles are 1 x 10 per unit volume of 1 mL of the composition. 8 Pieces up to 1 x 10 13 It can be included in the content of dogs.

[0042] In this specification, the term 'extracellular vesicle' refers to a small membrane-bound vesicle secreted from various cells, having a diameter in the range of approximately 20 to 1,000 nm, and representative extracellular vesicles may have a size of about 50 to 300 nm or 50 to 200 nm. In particular, extracellular vesicles derived from red cabbage form particles of a uniform size of approximately 50 to 200 nm in the form of a double lipid membrane.

[0043]

[0044] One aspect of the present invention is,

[0045] Step of preparing plant juice;

[0046] A step of collecting the supernatant after centrifuging the above extract;

[0047] A step of obtaining a filtrate by performing ultrafiltration on the above supernatant; and

[0048] The method for isolating plant-derived extracellular vesicles includes sequentially the step of purifying the above-mentioned filtrate.

[0049] The above centrifugation may be performed at 4,000 to 30,000 g. Below this range, the separation efficiency or ability of extracellular vesicles may be poor, and above this range, there may be a risk of damage to the extracellular vesicles.

[0050] In particular, the step of collecting the supernatant after centrifugation of the above-mentioned extract may be repeated two or more times, characterized by gradually increasing the gravitational acceleration during centrifugation. For example, the first centrifugation may be performed at 4,000 to 10,000 g, and the second centrifugation may be performed at 15,000 to 30,000 g. Through such a multi-stage centrifugation method proceeding from high speed to ultra-high speed, separation efficiency relative to time and reduced damage to extracellular vesicles can be improved, and stability can be enhanced, making it suitable for mass production.

[0051] The above ultrafiltration preferably has a cut-off value of 50 to 150 kDa, more preferably has a cut-off value of 80 to 120 kDa, and even more preferably has a value of around 100 kDa. At the above range of cut-off values, the centrifugation method can increase separation efficiency relative to time, making it suitable for mass production.

[0052] The above purification step may utilize various purification technologies of the prior art, but it is preferable to perform it using activated carbon. The prior art polymer precipitation method has a very high impurity concentration and may cause environmental pollution problems due to the use of PEG (polyethylene glycol), and the separation method using size exclusion chromatography (SEC) is too time-consuming, which limits its commercial application, particularly mass production.

[0053] In particular, the inventors have established a purification process suitable for mass production using a purification method with activated carbon, which involves mixing the activated carbon and the material to be purified for a predetermined period of time. The stirring time is preferably within 10 minutes to 2 hours, more preferably within 30 minutes to 90 minutes, and even more preferably within the range of approximately 60 minutes. If the stirring time is too short, it is insufficient for purification, and if it is too long, the purification efficiency relative to time is not high, so it may not be suitable for mass production. At this time, the stirring rpm may be in the range of approximately 35 to 40 rpm.

[0054] The activated carbon used in the present invention may be used in an amount of 10 to 25% (by weight), and is preferably approximately 15% (by weight). Below this range, purification may not be effective or there may be no effect of improving the properties, and above this range, there may be a risk of a problem where the yield of extracellular vesicles decreases.

[0055] In the present invention, the types of plants are not limited and may be used for any type of plant having a cell wall, but examples of suitable plants that can be used in the separation method of the present invention include cruciferous plants such as cabbage, Chinese cabbage, kohlrabi, kale, broccoli, radish, rapeseed, Brussels sprouts, red cabbage, and cauliflower.

[0056] The separation method of the present invention can extract extracellular vesicles with a high yield relative to time. That is, a composition containing a high concentration of extracellular vesicles can be obtained in a relatively short time, for example, based on the volume of the composition, at least 1 x 10 per unit volume of 1 mL of the composition 8 particles or more up to 1 x 10 13 It may contain particles in an amount less than 1 x 10 per unit volume of 1 mL of the composition, and more specifically, 1 x 10 10 More than 1 x 10 11 It may contain more than one. The fact that such a high concentration of extracellular vesicles can be obtained without a separate reduced-pressure concentration process indicates that the separation method of the present invention is highly suitable for mass production.

[0057]

[0058] Another aspect of the present invention is,

[0059] This is a cosmetic composition comprising a plant-derived extracellular vesicle obtained by the separation method of the present invention.

[0060] Here, the extracellular vesicle is 1 x 10 per unit volume of 1 mL of the composition, based on the volume of the composition. 8 ~ 1 x 10 13 It may be included in the particle content.

[0061] Preferably, the plant may be derived from red cabbage, and the extracellular endoplasmic reticulum is 1 x 10 per unit volume of 1 mL of the composition, based on the volume of the composition. 10 ~ 1 x 10 12 particles, more preferably 1 x 10 11 ~ 1 x 10 12 It may be included in a high content of particles. However, the possibility that the content may decrease slightly during storage from the initial manufacturing date should also be fully considered. For example, when stored for 6 months to over 1 year, approximately 10% compared to the content at the time of initial storage2 A decrease in content may occur within the internal and external ranges.

[0062]

[0063] Another aspect of the present invention is,

[0064] It is a cosmetic composition containing extracellular vesicles derived from red cabbage as an active ingredient, and having antioxidant, anti-inflammatory, or skin-soothing effects.

[0065] Extracellular vesicles derived from red cabbage allow for good intracellular penetration, and since the delivered extracellular vesicles have beneficial effects on the skin, such as antioxidant, anti-inflammatory, and skin-soothing properties, they can be usefully utilized in cosmetic compositions. A more detailed understanding of this can be achieved through the examples and test examples described below.

[0066]

[0067] The present invention will be explained in more detail below through embodiments and experimental examples. However, it should be clarified that the following embodiments are for the purpose of explaining the invention in detail only and are not intended to limit the scope of the rights.

[0068]

[0069] Examples

[0070] Example 1: Preparation of extracellular vesicles derived from red cabbage

[0071] 1. Washing and juicing red cabbage

[0072] Red cabbage was washed three times with distilled water to remove dust, soil, and pesticides. Juice was extracted from the washed red cabbage using a juicer. The juice extracted from the red cabbage was filtered through a cotton bag to remove large particles. For filtration, a cotton bag designed to allow only chunks smaller than 1 mm in width and 1 mm in length to pass through was used to effectively remove large substances.

[0073] 2. Removal of protein and cell wall

[0074] Centrifugation was performed to remove proteins and cell wall components from red cabbage juice from which large particles had been removed. After centrifugation at 8,000×g for 1 hour at 4℃, only the supernatant was collected. The supernatant was centrifuged again at 20,000×g for 1 hour at 4℃ to obtain a supernatant containing extracellular endoplasmic reticulum.

[0075] 3. Isolation and Purification of Extracellular Vesicles

[0076] To isolate extracellular vesicles, the centrifuged supernatant was subjected to an additional membrane separation process using an ultrafiltration (UF). In this process, a TFF (stationary flow filtration) membrane filter with a molecular weight cutoff limit of 100 kDa was used to remove other impurity particles smaller than the pores of the filter, thereby isolating the extracellular vesicles.

[0077] Subsequently, the extracellular vesicles obtained as described above were purified by using 15% (by weight) of activated carbon and stirring at 20–30°C for 1–1.5 hours. Finally, extracellular vesicles with a diameter in the range of 20 to 200 nm could be prepared.

[0078]

[0079] Example 2: Preparation of a cream formulation using a cosmetic composition containing extracellular vesicles derived from red cabbage

[0080] A cosmetic composition containing extracellular vesicles derived from red cabbage was prepared as a cream formulation with the composition of Table 1 below.

[0081] Phase Component Example 2 (% by weight) Comparative Example 1 (% by weight) AWaterTo 100To 100Disodium EDTA0.020.02Glycerin55Butylene glycol5.55.5Cabopol 9800.30.3BCetearyl Olivate1.51.5Sorbitan Olivate0.50.5Cetearyl Alcohol2.52.5Butyrospermum Parkii Butter33Caprylic Capric Triglyceride55Coco Caprylate Caprate22Undecane, Tridecane33CTromethamine0.280.28DSodium Acrylate / Sodium Acryloyldimethyl Taurate Copolymer, Polyisobutene, Caprylyl Capryl Glucoside0.50.5EVinyl Dimethicone11FDerived from red cabbage Extracellular endoplasmic reticulum (Example 1) 5-1,2 Hexanediol 1.51.5

[0082]

[0083] Test example

[0084] Test Example 1: Confirmation of concentration and particle size of extracellular vesicles derived from red cabbage

[0085] Nanoparticle tracking analyzer (NTA) measurements were performed to confirm the concentration and particle size of the extracellular vesicles derived from red cabbage in Example 1. The measurement results are shown in Figures 1 and 2.

[0086] As a result, the concentration was 6.4 x 10 11 It was confirmed that the particle size was 124 nm, with particles / mL.

[0087]

[0088] Test Example 2: Confirmation of NO production inhibitory effect of cosmetic composition containing extracellular vesicles derived from red cabbage

[0089] The skin soothing effect of the cosmetic composition (cream formulation) containing the extracellular vesicles derived from red cabbage of Example 2 was confirmed as follows.

[0090] RAW264.7 cells 1.5 x 10 5 The cells were prepared at a concentration of cells / ml, inoculated into a 24-well plate in DMEM medium, and cultured for 24 hours. Subsequently, fresh medium containing the test substance and LPS (1 μg / ml) was added, and the cells were cultured for an additional 24 hours. After culture, 100 μl of the cell supernatant and 100 μl of Griess reagent were added to a 96-well plate and reacted for 15 minutes. After the reaction was complete, an ELISA assay was performed according to the manufacturer's manual to determine NO - 2. The amount produced was measured. The measurement results are as shown in Figure 3.

[0091] As a result, it was confirmed that NO production was inhibited in a concentration-dependent manner upon treatment with extracellular vesicles derived from red cabbage.

[0092]

[0093] Test Example 3: Confirmation of the inhibitory effect of a cosmetic composition containing extracellular vesicles derived from red cabbage on IL-6 and IL-1β production

[0094] The skin soothing effect of the cosmetic composition (cream formulation) containing the extracellular vesicles derived from red cabbage of Example 2 was confirmed as follows.

[0095] RAW264.7 cells 1.5 x 10 5 Cells were inoculated into a 24-well plate at a rate of cells / well and cultured for 24 hours. Subsequently, a medium containing the test substance and LPS (1 μg / ml) was added simultaneously, and the cells were cultured for an additional 24 hours. After culture, the cytokine (IL-6, IL-1β) content in the cell culture medium was measured using an inflammatory cytokine ELISA kit according to the manufacturer's manual. The measurement results are shown in Figures 4 and 5.

[0096] As a result, it was confirmed that treatment with extracellular vesicles derived from red cabbage inhibited the production of IL-6 and IL-1β in a concentration-dependent manner.

[0097]

[0098] Test Example 4: Measurement of soothing of physically irritated skin using a cream formulation applied with a cosmetic composition containing extracellular vesicles derived from red cabbage.

[0099] All 23 subjects who completed this study were female, with an average age of 44.304 years. The selected subjects had no specific skin symptoms and no history of diseases or medication use that could affect the study.

[0100] The test product was used by dividing one side of the subject's face into sections (width x height, 2 cm x 2 cm) starting from the left, with the test product, negative control product, and unapplied area, and applying the test product and negative control product at a rate of 2 µl / cm².

[0101] The product was used once. For instrumental evaluation, the test subjects rested for 30 minutes in a waiting room under constant temperature and humidity conditions of 20–24°C and 40–60% humidity to allow their skin surface temperature and humidity to adapt to the environment of the measurement space, and fluid intake was restricted during the resting period. For objective measurement, one researcher performed the measurements, and the same area was measured at each measurement.

[0102] Skin damage was induced on the same right forearm of the subject by repeating tape stripping (TS) 20 times using a special film (keratin tape, Air D&C & C-Beauty, Korea).

[0103] To soothe physically irritated skin, the same right forearm of the subjects was photographed before and after the use of the test product using an Antera 3D CS (Miravex Ltd., Ireland). The analysis parameter was the a* value, which indicates skin redness; a decrease in the value after use compared to after TS indicates an effect in soothing physically irritated skin. The measurement results are shown in Table 2, comparing the use of the test product, the control product, and the non-graphical product.

[0104] (Mean±SD) Category a* Test Product Control Product Uncoated TS Before 7.991±5.27 17.903±4.66 77.911±4.858 TS After 29.428±7.660 29.598±7.921 29.260±9.059 Immediately After Use 12.287±7.664 17.500±9.289 23.057±10.002 Improvement Rate (%) TS After - Immediately After Use 58.247 40.874 21.200 Within-group Comparison p-value Within-subject Effect Test 0.000**0.000**0.000**TS Before - TS After 0.000**0.000**0.000**TS After - Immediately After Use 0.000**0.000**0.000**Test Product - Uncoated Test Product - Control Product Control Product - Comparison between Unapplied Group p-value Within-subject effect test 0.000**0.000**0.000**TS Before - TS After 0.951 0.815 0.797TS After - Immediately after use 0.000**0.000**0.000**

[0105] ※ Improvement rate (%)= │(after-before)│ / before*100

[0106] ***: p<0.05 by repeated measures ANOVA, post hoc CI correction by Bonferroni.

[0107] Ŧ: p<0.05 by repeated measures ANOVA

[0108]

[0109] As a result, the test product showed a statistically significant increase after TS compared to before TS compared to the use of the control product and no application, and a statistically significant improvement immediately after use compared to after TS. In other words, it was confirmed that a single application helps soothe skin irritated by physical stimulation.

[0110] The present invention can efficiently separate and purify plant-derived extracellular vesicles with high purity and high quality. Since the plant-derived extracellular vesicles produced by the method of the present invention have good intracellular permeability and the delivered extracellular vesicles have beneficial effects on the skin, such as antioxidant, anti-inflammatory, and skin soothing effects, they can be usefully utilized in various industrial fields, such as the manufacture of health functional foods, cosmetics, quasi-drugs, or pharmaceutical compositions for improving skin condition.

Claims

1. Step of preparing plant juice; A step of collecting the supernatant after centrifuging the above extract; A step of obtaining a filtrate by performing ultrafiltration on the above supernatant; and A method for isolating plant-derived extracellular vesicles, comprising sequentially the step of purifying the above-mentioned filtrate.

2. A method for isolating plant-derived extracellular vesicles according to claim 1, characterized in that the centrifugation is performed at 4,000 to 30,000 g.

3. A method for isolating plant-derived extracellular vesicles, wherein, in Claim 1, the step of collecting the supernatant after centrifuging the extract is repeated two or more times, and the gravitational acceleration during centrifugation is gradually increased.

4. A method for isolating plant-derived extracellular vesicles according to claim 3, characterized in that the first centrifugation is performed at 4,000 to 10,000 g and the second centrifugation is performed at 15,000 to 30,000 g.

5. A method for isolating plant-derived extracellular vesicles according to Claim 1, characterized in that the ultrafiltration has a cut-off value of 50 to 150 kDa.

6. A method for isolating plant-derived extracellular vesicles according to Claim 1, wherein the purification step uses activated carbon.

7. A method for isolating plant-derived extracellular vesicles according to Claim 1, characterized in that the plant is red cabbage.

8. A cosmetic composition comprising a plant-derived extracellular vesicle obtained by the separation method of any one of claims 1 to 7.

9. In claim 8, the extracellular vesicle is 1 x 10 per unit volume of 1 mL of the composition, based on the volume of the composition. 8 ~ 1 x 10 13 A cosmetic composition characterized by containing particles.

10. A cosmetic composition characterized by containing extracellular vesicles derived from red cabbage as an active ingredient and having antioxidant efficacy.

11. A cosmetic composition characterized by containing extracellular vesicles derived from red cabbage as an active ingredient and having anti-inflammatory efficacy.

12. A cosmetic composition characterized by containing extracellular vesicles derived from red cabbage as an active ingredient and having a skin soothing effect.

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